Recent data from our laboratory have defined a novel nuclear action of insulin, which is inhibition of nuclear phosphatase PP-2A activity. This inhibition of nuclear phosphatase activity is associated with enhanced phosphorylaiton of cyclic AMP response element binding protein (CREB). This led us to the following hypothesis; Insulin signaling leads to a transient inhibition of nuclear PP-2A activity which results in enhanced phosphorylation state of CREB and other transcription factors. This alteration in transcription factor phosphorylation is one mechanism of insulin mediated gene regulation. To explore this hypothesis, we will address the following specific aims: 1. To identify the intermediate steps of the insulin signaling cascade responsible for the inhibition of nuclear PP-2A. A. These experiments will use currently available cell lines containing mutant insulin receptors, pharmacologic inhibitors, and a cell line deficient in IRS-1, to determine the receptor-related signaling elements essential to PP-2A regulation. B. To establish the key cytoplasmic components required for PP-2A regulation in vitro and in vivo. These experiments will employ a cell free nuclear incubation system and an inducible expression system for dominant positive and negative Ras, Raf-1, and MEK. 2. To establish the insulin mediated changes in the phosphorylation state of TF's and nuclear regulatory proteins, and its impact upon transcriptional regulation. A. Examine the effects of insulin upon the phosphorylation states and rates of dephosphorylation of CREB, CREB-BP, c\Jun, and c-Fos. These studies will use in vivo [32P]-labeled cells treated with and without insulin. Antibody immunoprecipitation and assessment of the phosphorylation state will establish changes in phosphorylation state in response to insulin. B. Examine the effects of insulin upon the transcriptional transactivating activity of CREB, c-Fos, and c-Jun using in vitro transcription assays. These studies will employ a Gal4 reporter system activated by chimeric Ga14/transcription factor fusion proteins.